Nucleostemin基因荧光真核表达载体构建及表达特性

Construction and expression of nucleostemin gene fluorescent eukaryotic cell expression vector

目的:构建Nucleostemin(NS)基因真核表达载体,并在COS-7细胞中表达。方法:设计引物在肺癌细胞株(LTEP-a-2)中扩增NS全长cDNA片段,插入pMD-18T质粒中得pMD-18T-NS载体,并测序;再将pMD-18T-NS质粒经酶切后导入真核表达载体pcDNA3.1(+)-GFP质粒中,构建pcDNA3.1(+)-GFP-NS载体,并检测其真核表达情况。结果:经RT-PCR扩增得到1650bp的全长NScDNA片段,酶切及测序后鉴定证明pMD-18T-NS构建成功。pcDNA3.1(+)-G-FP-NS载体转染COS-7细胞经蛋白印迹及激光共聚焦显微镜检测显示,该GFP-N-S融合基因在COS-7细胞中获得较好表达。转染后见该基因定位于核仁,细胞逐渐失去黏附贴壁的特性,细胞的增殖受阻;稳定表达(4～20周)后,细胞形态发生改变,体积增大,核增多,细胞成瘤细胞样改变。结论:成功构建pcDNA3.1(+)-GF-P-NS真核表达载体,并在COS-7细胞中有效表达;NS基因在COS-7发生瘤样改变过程中发挥着重要作用,可能与肿瘤发生密切相关。

OBJECTIVE：To construct nucleostemin（NS）gene fluorescent eukaryotic cell expression vector,and detect its expression in a eukaryotic cell COS-7.METHODS：The full-length NS cDNA fragment of lung cancer cell line（LTEP-a-2）was synthesized by RT-PCR,the fragment was linked up with pMD-18T vector by co-adhesive end restriction,named pMD-18T-NS vector and sequenced,and then NS cDNA fragment obtained from pMD-18T-NS vector was ligated with pcDNA3.1（＋）-GFP to construct fluorescent eukaryotic cell expression vector pcDNA3.1（＋）-GFP-NS,after being identified and expressed in COS-7 cells.RESULTS：A 1 650 bp fragment of the full-length NS cDNA was amplified by RT-PCR,using digestion and sequencing to prove successful construction of pMD-18T-NS vector.The results revealed that in COS-7 cells the GFP-NS fusion gene was expressed better after pcDNA3.1（＋）-GFP-NS vector transfect COS-7 cells.By Western blot and laser scanning confocal microscope,it was seen that the transfected gene located in the nucleolus,and the cells gradually lose the characteristics of adherent adhesion,and cell proliferation blocked.A tumor-like change,such as the changes in cell morphology,increased volume and nuclear number,was find after stable expression of NS（4-20 weeks）.CONCLUSIONS：The pcDNA3.1（＋）-GFP-NS eukaryotic expression vector is constructed successfully and effectively expressed in the COS-7 cells.NS might be one of the factors playing important roles during tumour genesis.